RESUMEN
Malignant pleural mesothelioma, a tumor arising from the membrane covering the lungs and the inner side of the ribs, is a cancer in which genetic alterations of genes encoding proteins that act on or are part of the Hippo-YAP1 signaling pathway are frequent. Dysfunctional Hippo signaling may result in aberrant activation of the transcriptional coactivator protein YAP1, which binds to and activates transcription factors of the TEAD family. Recent studies have associated elevated YAP1 protein activity with a poor prognosis of malignant mesothelioma and its resistance to current therapies, but its role in tumor maintenance is unclear. In this study, we investigate the dependence of malignant mesothelioma on YAP1 signaling to maintain fully established tumors in vivo. We show that downregulation of YAP1 in a dysfunctional Hippo genetic background results in the inhibition of YAP1/TEAD-dependent gene expression, the induction of apoptosis, and the inhibition of tumor cell growth in vitro. The conditional downregulation of YAP1 in established tumor xenografts leads to the inhibition of YAP1-dependent gene transcription and eventually tumor regression. This effect is only seen in the YAP1-activated MSTO-211H mesothelioma xenograft model, but not in the Hippo-independent HCT116 colon cancer xenograft model. Our data demonstrate that, in the context of a Hippo pathway mutated background, YAP1 activity alone is enough to maintain the growth of established tumors in vivo, thus validating the concept of inhibiting the activated YAP1-TEAD complex for the treatment of malignant pleural mesothelioma patients.
Asunto(s)
Mesotelioma Maligno , Mesotelioma , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Mesotelioma/patología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Señalizadoras YAPRESUMEN
E-prostanoid receptor subtype 2 (EP2) agonists are currently under clinical development as hypotensive agents for the treatment of ocular hypertension. However, the effects of EP2 receptor agonists on trabecular meshwork (TM) alterations leading to primary open-angle glaucoma (POAG) are still unknown. Here, we evaluated whether EP2 receptor activation exhibits protective functions on TM cell death induced by endoplasmic reticulum (ER) stress. We show that the EP2 receptor agonist butaprost protects TM cell death mediated by the ER stress inducer tunicamycin through a cyclic AMP (cAMP)-dependent mechanism, but independent of the classical cAMP sensors, protein kinase A and exchange proteins activated by cAMP. The ER stress-induced intrinsic apoptosis inhibited by the EP2 receptor agonist was correlated with a decreased accumulation of the cellular stress sensor p53. In addition, p53 down-regulation was associated with inhibition of its transcriptional activity, which led to decreased expression of the pro-apoptotic p53-upregulated modulator of apoptosis (PUMA). The stabilization of p53 by nutlin-3a abolished butaprost-mediated cell death protection. In conclusion, we showed that EP2 receptor activation protects against ER stress-dependent mitochondrial apoptosis through down-regulation of p53. The specific inhibition of this pathway could reduce TM alterations observed in POAG patients.
Asunto(s)
Apoptosis , Citoprotección , Regulación hacia Abajo , Estrés del Retículo Endoplásmico , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal , Malla Trabecular/patología , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Alprostadil/análogos & derivados , Alprostadil/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocromos c/metabolismo , Citoprotección/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción CHOP/metabolismo , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Microbial natural products are a rich source of bioactive molecules to serve as drug leads and/or biological tools. We investigated a little-explored myxobacterial genus, Nannocystis sp., and discovered a novel 21-membered macrocyclic scaffold that is composed of a tripeptide and a polyketide part with an epoxyamide moiety. The relative and absolute configurations of the nine stereocenters was determined by NMR spectroscopy, molecular dynamics calculations, chemical degradation, and X-ray crystallography. The compound, named nannocystin A (1), was found to inhibit cell proliferation at low nanomolar concentrations through the early induction of apoptosis. The mode of action of 1 could not be matched to that of standard drugs by transcriptional profiling and biochemical experiments. An initial investigation of the structure-activity relationship based on seven analogues demonstrated the importance of the epoxide moiety for high activity.
Asunto(s)
Antifúngicos/química , Antineoplásicos/química , Productos Biológicos/farmacología , Proliferación Celular/efectos de los fármacos , Compuestos Macrocíclicos/farmacología , Myxococcales/fisiología , Antifúngicos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Productos Biológicos/química , Candida albicans/efectos de los fármacos , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Compuestos Macrocíclicos/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The bengamides, sponge-derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M.â virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M.â xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half-life in mice and antitumor efficacy in a melanoma mouse model.
Asunto(s)
Azepinas/metabolismo , Productos Biológicos/metabolismo , Biología Marina , Myxococcales/metabolismo , Poríferos/metabolismo , Animales , Área Bajo la Curva , Azepinas/farmacocinética , Azepinas/farmacología , Productos Biológicos/farmacocinética , Productos Biológicos/farmacología , Femenino , Semivida , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Relación Estructura-ActividadRESUMEN
The TGFß signaling mediator SMAD4 is frequently mutated or deleted in colorectal and pancreatic cancers. SMAD4 acts as a tumor suppressor and its loss is associated with poorer patient outcomes. The purpose of this study was to find synthetic lethal interactions with SMAD4 deficiency to find novel therapeutic strategies for the treatment of patients with SMAD4-deficient colorectal or pancreatic cancers. Using pooled lentiviral single-guide RNA libraries, we conducted genome-wide loss-of-function screens in Cas9-expressing colorectal and pancreatic cancer cells harboring altered or wild-type SMAD4. The small GTPase protein RAB10 was identified and validated as a susceptibility gene in SMAD4-altered colorectal and pancreatic cancer cells. Rescue assays showed that RAB10 reintroduction reversed the antiproliferative effects of RAB10 knockout in SMAD4-negative cell lines. Further investigation is necessary to shed light on the mechanism by which RAB10 inhibition decreases cell proliferation of SMAD4-negative cells. Significance: This study identified and validated RAB10 as new synthetic lethal gene with SMAD4. This was achieved by conducting a whole-genome CRISPR screens in different colorectal and pancreatic cell lines. A future RAB10 inhibitors could correspond to a new therapeutic solution for patients with cancer with SMAD4 deletion.
Asunto(s)
Neoplasias Colorrectales , Neoplasias Pancreáticas , Humanos , Línea Celular Tumoral , Genes Letales , Neoplasias Pancreáticas/genética , Neoplasias Colorrectales/genética , Proteína Smad4/genética , Neoplasias PancreáticasRESUMEN
KRASG12C is one of the most common mutations detected in non-small cell lung cancer (NSCLC) patients, and it is a marker of poor prognosis. The first FDA-approved KRASG12C inhibitors, sotorasib and adagrasib, have been an enormous breakthrough for patients with KRASG12C mutant NSCLC; however, resistance to therapy is emerging. The transcriptional coactivators YAP1/TAZ and the family of transcription factors TEAD1-4 are the downstream effectors of the Hippo pathway and regulate essential cellular processes such as cell proliferation and cell survival. YAP1/TAZ-TEAD activity has further been implicated as a mechanism of resistance to targeted therapies. Here, we investigate the effect of combining TEAD inhibitors with KRASG12C inhibitors in KRASG12C mutant NSCLC tumor models. We show that TEAD inhibitors, while being inactive as single agents in KRASG12C-driven NSCLC cells, enhance KRASG12C inhibitor-mediated anti-tumor efficacy in vitro and in vivo. Mechanistically, the dual inhibition of KRASG12C and TEAD results in the downregulation of MYC and E2F signatures and in the alteration of the G2/M checkpoint, converging in an increase in G1 and a decrease in G2/M cell cycle phases. Our data suggest that the co-inhibition of KRASG12C and TEAD leads to a specific dual cell cycle arrest in KRASG12C NSCLC cells.
RESUMEN
KRAS genes belong to the most frequently mutated family of oncogenes in cancer. The G12C mutation, found in a third of lung, half of colorectal and pancreatic cancer cases, is believed to be responsible for a substantial number of cancer deaths. For 30 years, KRAS has been the subject of extensive drug-targeting efforts aimed at targeting KRAS protein itself, but also its post-translational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. So far, most KRAS targeting strategies have failed, and there are no KRAS-specific drugs available. However, clinical candidates targeting the KRAS G12C protein have recently been developed. MRTX849 and recently approved Sotorasib are covalent binders targeting the mutated cysteine 12, occupying Switch II pocket.Herein, we describe two fragment screening drug discovery campaigns that led to the identification of binding pockets on the KRAS G12C surface that have not previously been described. One screen focused on non-covalent binders to KRAS G12C, the other on covalent binders.
Asunto(s)
Antineoplásicos , Neoplasias , Acetonitrilos/uso terapéutico , Antineoplásicos/uso terapéutico , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Piperazinas , Proteínas Proto-Oncogénicas p21(ras)/genética , PirimidinasRESUMEN
Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAFV600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento , Ratones , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
Primary treatment for estrogen receptor-positive (ER+) breast cancer is endocrine therapy. However, substantial evidence indicates a continued role for ER signaling in tumor progression. Selective estrogen receptor degraders (SERD), such as fulvestrant, induce effective ER signaling inhibition, although clinical studies with fulvestrant report insufficient blockade of ER signaling, possibly due to suboptimal pharmaceutical properties. Furthermore, activating mutations in the ER have emerged as a resistance mechanism to current endocrine therapies. New oral SERDs with improved drug properties are under clinical investigation, but the biological profile that could translate to improved therapeutic benefit remains unclear. Here, we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradation activities against both wild-type and mutant Y537S ER. Driven by its fluoropropyl pyrrolidinyl side chain, SAR439859 has demonstrated broader and superior ER antagonist and degrader activities across a large panel of ER+ cells, compared with other SERDs characterized by a cinnamic acid side chain, including improved inhibition of ER signaling and tumor cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ breast cancer models, including MCF7-ESR1 wild-type and mutant-Y537S mouse tumors, and HCI013, a patient-derived tamoxifen-resistant xenograft tumor. These findings indicate that SAR439859 may provide therapeutic benefit to patients with ER+ breast cancer, including those who have resistance to endocrine therapy with both wild-type and mutant ER.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Receptores de Estrógenos/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , RatonesRESUMEN
Controversy exists surrounding whether heterogeneous disruption of the blood-brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. In vitro efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both in vitro and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. Mol Cancer Ther; 17(9); 1893-901. ©2018 AACR.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Indoles/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Indoles/farmacocinética , Masculino , Ratones , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Compuestos de Espiro/farmacocinética , Análisis de Supervivencia , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
TP53 also known as p53 is a tumor suppressor gene mutated in a variety of cancers. P53 is involved in cell cycle, apoptosis and DNA repair mechanisms and is thus tightly controlled by many regulators. Recently, strategies to treat cancer have focused on the development of MDM2 antagonists to induce p53 stabilization and restore cell death in p53 non-mutated cancers. However, some of these molecules display adverse effects in patients including induction of thrombocytopenia. In the present study, we have explored the effect of SAR405838 not only on human megakaryopoiesis but also more generally on hematopoiesis. We compared its effect to MI-219 and Nutlin, which are less potent MDM2 antagonists than SAR405838. We found that all these compounds induce a deleterious effect on all types of hematopoietic progenitors, as well as on erythroid and megakaryocytic differentiation. Moreover, they inhibit both early and late stages of megakaryopoiesis including ploidization and proplatelet formation. In conclusion, MDM2 antagonists induced a major hematopoietic defect in vitro as well as an inhibition of all stages of megakaryopoiesis that may account for in vivo thrombocytopenia observed in treated patients.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Indoles/toxicidad , Compuestos de Espiro/toxicidad , Trombopoyesis/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Antígenos CD34/metabolismo , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Imidazoles/farmacología , Indoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Compuestos de Espiro/farmacología , Trombocitopenia/sangre , Trombocitopenia/inducido químicamente , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PURPOSE: Dedifferentiated liposarcoma (DDLPS) is an aggressive malignancy that can recur locally or disseminate even after multidisciplinary care. Genetically amplified and expressed MDM2, often referred to as a "hallmark" of DDLPS, mostly sustains a wild-type p53 genotype, substantiating the MDM2:p53 axis as a potential therapeutic target for DDLPS. Here, we report on the preclinical effects of SAR405838, a novel and highly selective MDM2 small-molecule inhibitor, in both in vitro and in vivo DDLPS models. EXPERIMENTAL DESIGN: The therapeutic effectiveness of SAR405838 was compared with the known MDM2 antagonists Nutlin-3a and MI-219. The effects of MDM2 inhibition were assessed in both in vitro and in vivo. In vitro and in vivo microarray analyses were performed to assess differentially expressed genes induced by SAR405838, as well as the pathways that these modulated genes enriched. RESULTS: SAR405838 effectively stabilized p53 and activated the p53 pathway, resulting in abrogated cellular proliferation, cell-cycle arrest, and apoptosis. Similar results were observed with Nutlin-3a and MI-219; however, significantly higher concentrations were required. In vitro effectiveness of SAR405838 activity was recapitulated in DDLPS xenograft models where significant decreases in tumorigenicity were observed. Microarray analyses revealed genes enriching the p53 signaling pathway as well as genomic stability and DNA damage following SAR405838 treatment. CONCLUSIONS: SAR405838 is currently in early-phase clinical trials for a number of malignancies, including sarcoma, and our in vitro and in vivo results support its use as a potential therapeutic strategy for the treatment of DDLPS.
Asunto(s)
Indoles/administración & dosificación , Liposarcoma/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/genética , Compuestos de Espiro/administración & dosificación , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Imidazoles/administración & dosificación , Liposarcoma/genética , Liposarcoma/patología , Ratones , Análisis por Micromatrices , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Piperazinas/administración & dosificación , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Two clinical-stage anticancer drugs, the Bcl-2 inhibitor ABT-263, and the MDM2 inhibitor SAR405838 achieve complete tumor regression in animal models of leukemia but also induce acquired resistance. Elucidation of acquired resistance mechanisms and development of strategies to overcome the resistance are critical for their successful clinical development. EXPERIMENTAL DESIGN: We employed RS4;11 and MV4;11 cell lines, two acute leukemia models, to investigate acquired resistance mechanisms for both drugs in vitro and in vivo and evaluated several treatment regimens in xenograft mouse models to improve long-term, complete tumor regression. RESULTS: Resistance to either SAR405838 or ABT-263 (or its analogue ABT-737) develops in acute leukemia models in vitro and in vivo. RS4;11 and MV4;11 tumors treated with SAR405838 acquire resistance to the drug by mutation of the TP53 gene or compromise of p53 protein function. RS4;11 tumors treated with either ABT-263 or ABT-737 acquire resistance primarily through downregulation of BAX but not BAK. When acute leukemia cells become highly resistant to the MDM2 inhibitor, they retain their sensitivity to the Bcl-2 inhibitors, or vice versa. Certain sequential or combination treatment of SAR405838 and ABT-263 can achieve longer-term tumor regression than treatment with either agent alone. CONCLUSIONS: Our study provides new insights into the mechanisms of acquired resistance of Bcl-2 and MDM2 inhibitors in acute leukemia models and suggests that certain sequential or combination treatment of these two distinct classes of apoptosis-inducing agents should be tested as new treatment strategies for acute leukemia in the clinic.
Asunto(s)
Leucemia/tratamiento farmacológico , Leucemia/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/administración & dosificación , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Indoles/administración & dosificación , Leucemia/patología , Ratones , Nitrofenoles/administración & dosificación , Piperazinas/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Compuestos de Espiro/administración & dosificación , Sulfonamidas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SAR405838 is a potent and specific MDM2 inhibitor currently being evaluated in Phase I clinical trials for the treatment of human cancer. Using the SJSA-1 osteosarcoma cell line which harbors an amplified MDM2 gene and wild-type p53, we have investigated the acquired resistance mechanisms both in vitro and in vivo to SAR405838. Treatment of SJSA-1 cells with SAR405838 in vitro leads to dose-dependent cell growth inhibition, cell cycle arrest and robust apoptosis. However, prolonged treatment of SJSA-1 cells in vitro with SAR405838 results in profound acquired resistance to the drug. Analysis of in vitro-derived resistant cell lines showed that p53 is mutated in the DNA binding domain and can no longer be activated by SAR405838. Treatment of the parental SJSA-1 xenograft tumors with SAR405838 in mice yields rapid tumor regression but the tumors eventually regrow. Culturing the regrown tumors established a number of sublines, which showed only modest (3-5 times) loss of sensitivity to SAR405838 in vitro. Sequencing of the p53 showed that it retains its wild-type status in these in vivo sublines, with the exception of one subline, which harbors a single heterozygous C176F p53 mutation. Using xenograft models of two in vivo derived sublines, which has either wild-type p53 or p53 containing a single heterozygous C176F mutation, we showed that while SAR405838 effectively achieves partial tumor regression in these models, it no longer induces complete tumor regression and tumors resume growth once the treatment is stopped. Harvesting and culturing tumors obtained from a prolonged treatment with SAR405838 in mice established additional in vivo sublines, which all contain a single heterozygous C176F mutation with no additional p53 mutation detected. Interestingly, SAR405838 can still effectively activate p53 in all sublines containing a single heterozygous C176F mutation, with a moderately reduced potency as compared to that in the parental cell line. Consistently, SAR405838 is 3-5 times less effective in all the in vivo derived sublines containing a single heterozygous C176F p53 mutation than in the SJSA-1 parental cell line in assays of cell growth and apoptosis. Computational modeling suggested that a p53 tetramer containing two wild-type p53 molecules and two C176F mutated molecules can maintain the structural stability and interactions with DNA by formation of additional hydrophobic and cation-π interactions which compensate for the loss of sulphur-zinc coordination. Our data thus show that SJSA-1 tumor cells acquire very different levels of resistance in vitro and in vivo to the MDM2 inhibitor SAR405838. Our present study may have a significant implication for the investigation of resistant mechanisms for other classes of anticancer drugs.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Indoles/farmacología , Osteosarcoma , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Sustitución de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Humanos , Ratones , Ratones SCID , Mutación Missense , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Blocking the oncoprotein murine double minute 2 (MDM2)-p53 protein-protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small-molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301), that has been advanced into phase I clinical trials. SAR405838 binds to MDM2 with K(i) = 0.88 nmol/L and has high specificity over other proteins. A cocrystal structure of the SAR405838:MDM2 complex shows that, in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer, and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional upregulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53.
Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Compuestos de Espiro/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Puntos de Control del Ciclo Celular , Proliferación Celular/efectos de los fármacos , Perros , Estabilidad de Medicamentos , Células HCT116 , Humanos , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Inducción de Remisión , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Insulin-like growth factor 1 receptor (IGF-1R) is overexpressed in many tumours and contributes to tumourigenicity, cell proliferation, metastasis and resistance, thus representing a promising therapeutic target. The human IGF-1R antagonistic monoclonal antibody EM164 (murine AVE1642) has shown activity in adult cancers and is being evaluated in patients with advanced malignancies. We investigated the EM164 for its therapeutic potential against childhood neuroblastoma. EM164 at 0.07, 0.7 and 7 µg/mL exhibited anti-proliferative activity against all nine cell lines tested in (3)H-thymidine incorporation assay in vitro. Cell proliferation after EM164 exposure ranged between 24% and 80% compared to controls. Sensitivity was independent from culture serum conditions, intensity of IGF-1R expression and IGF-II secretion, although associated with inhibition of AKT activation. In vivo, EM164 administered intravenously at 40 mg/kg twice weekly for 4 weeks yielded significant tumour growth delays (TGD) of 13.4d in advanced stage IGR-N91 and 12.9 d in SK-N-AS tumours compared to controls (p = 0.02 and p = 0.0059, respectively). Simultaneous treatment of EM164 0.7 µg/mL and temozolomide resulted in enhanced activity in vitro. In vivo, treatment with temozolomide at the maximum tolerated dose (100mg/kg/d for 5 consecutive days) and EM164 yielded a significantly greater TGD of 29.1d (p<0.01) and two complete tumour regressions (CR) compared to 18.1d (p = ns) and one CR for EM164 alone and 16.1d (p = ns) for temozolomide alone. Our results demonstrate the potential of the anti-IGF-1R antibody alone and in combination with alkylating agents and support the therapeutic development of the AVE1642 for aggressive neuroblastoma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Alquilantes/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Antineoplásicos Alquilantes/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Femenino , Humanos , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Temozolomida , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.
Asunto(s)
Transformación Celular Neoplásica , Genes ras , Microtúbulos/efectos de los fármacos , Proteínas Oncogénicas/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-ets/metabolismo , Pirazoles/metabolismo , Factores de Transcripción/metabolismo , Actinas/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Ratones , Microtúbulos/metabolismo , Células 3T3 NIH , Proteínas Proto-Oncogénicas , Pirazoles/farmacologíaRESUMEN
Insulin-like growth factor 1 (IGF-1) is a well-known growth factor for myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for multiple myeloma treatment. In this study, we investigated the effect of the antagonistic anti-IGF-1R murineAVE1642 Ab (mAVE1642). We show that mAVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines. Since we have previously shown the functional relevance of CD45 expression in the growth of myeloma cells and the association of CD45-negative (CD45neg) status with a less favorable clinical outcome, both CD45-positive (CD45pos) and CD45neg myeloma cell lines were selected for our study. We found that mAVE1642 strongly inhibits the growth of CD45neg myeloma cell lines, leading to a G1 growth arrest, whereas it has almost no effect on the growth of CD45pos myeloma cell lines. Furthermore, mAVE1642 binding induced a significant reduction of IGF-1R expression. We next demonstrated that the overexpression of IGF-1R in the CD45pos myeloma cell line increased Akt phosphorylation but was not sufficient to sensitize these cells to mAVE1642. In contrast, we generated a stable CD45-silencing XG-1 cell line and showed that it became sensitive to mAVE1642. Thus, for the first time, we provided direct evidence that the expression of CD45 renders cells resistant to mAVE1642. Taken together, these results support that therapy directed against IGF-1R can be beneficial in treating CD45neg patients.